Plants with Modified Deoxyhypusine Synthase Genes

ABSTRACT

The invention relates to methods of producing a plant with delayed senescence comprising inducing at least one nucleotide deletion, insertion or substitution into at least one copy of a gene encoding deoxyhypusine synthase (DHS) in the plant, wherein the nucleotide deletion, insertion or substitution decreases the activity of DHS encoded by the gene in the plant. The invention also relates to plants produced by the methods and progeny thereof.

SEQUENCE LISTING SUBMISSION VIA EFS-WEB

A computer readable text file, entitled “SequenceListing.txt,” createdon or about Feb. 4, 2021 with a file size of about 596 kb contains thesequence listing for this application and is hereby incorporated byreference in its entirety.

BACKGROUND OF THE INVENTION

Senescence is the terminal phase of biological development in the lifeof a plant. It presages death and occurs at various levels of biologicalorganization including the whole plant, organs, flowers and fruit,tissues and individual cells.

The onset of senescence can be induced by different factors bothinternal and external. Senescence is a complex, highly regulateddevelopmental stage in the life of a plant or plant tissue, such asfruit, flowers and leaves. Senescence results in the coordinatedbreakdown of cell membranes and macromolecules and the subsequentmobilization of metabolites to other parts of the plant.

In addition to the programmed senescence which takes place during normalplant development, death of cells and tissues and ensuing remobilizationof metabolites occurs as a coordinated response to external,environmental factors. External factors that induce premature initiationof senescence, which is also referred to as necrosis or apoptosis,include environmental stresses such as temperature, drought, poor lightor nutrient supply, as well as pathogen attack. Plant tissues exposed toenvironmental stress also produce ethylene, commonly known as stressethylene [Buchanan-Wollaston (1997) J. Exp. Botany 48:181-199; Wright,M. (1974) Plant 120:63-69]. Ethylene is known to cause senescence insome plants.

Senescence is not a passive process, but, rather, is an activelyregulated process that involves coordinated expression of specificgenes. During senescence, the levels of total RNA decrease and theexpression of many genes is switched off [Bate et al. (1991) J. Exper.Botany 42:801-11; Hensel et al. (1993) The Plant Cell 5:553-64].However, there is increasing evidence that the senescence processdepends on de novo transcription of nuclear genes. For example,senescence is blocked by inhibitors of mRNA and protein synthesis andenucleation. Molecular studies using cDNA from senescing leaves andgreen leaves for in vitro translation experiments show a changed patternof leaf protein products in senescing leaves [Thomas et al. (1992) J.Plant Physiol. 139:403-12]. With the use of differential screening andsubtractive hybridization techniques, many cDNA clones representingsenescence-induced genes have been identified from a range of differentplants, including both monocots and dicots, such as Arabidopsis, maize,cucumber, asparagus, tomato, rice and potato. Identification of genesthat are expressed specifically during senescence is hard evidence ofthe requirement for de novo transcription for senescence to proceed.

The events that take place during senescence appear to be highlycoordinated to allow maximum use of the cellular components beforenecrosis and death occur. Complex interactions involving the perceptionof specific signals and the induction of cascades of gene expressionmust occur to regulate this process. Expression of genes encodingsenescence related proteins is probably regulated via common activatorproteins that are, in turn, activated directly or indirectly by hormonalsignals. Little is known about the mechanisms involved in the initialsignaling or subsequent co-ordination of the process.

Coordinated gene expression requires factors involved in transcriptionand translation, including initiation factors. Translation initiationfactor genes have been isolated and characterized in a variety oforganisms, including plants. Translation initiation factors can controlthe rate at which mRNA populations are moved out of the nucleus, therate at which they are associated with a ribosome and to some extent canaffect the stability of specific mRNAs. [Zuk et al. (1998) EMBO J.17:2914-2925]. Indeed, one such translation initiation factor, which isnot required for global translation activity, is believed to shuttlespecific subsets of mRNAs from the nucleus to the cytoplasm fortranslation [Jao et al. (2002) J. Cell. Biochem. 86:590-600; Wang et al.(2001) J. Biol. Chem. 276:17541-17549; Rosorius et al. (1999) J. CellSci. 112:2369-2380]. This translation factor is known as the eukaryoticinitiation factor 5A (eIF-5A), and is the only protein known to containthe amino acid hypusine [Park et al. (1988) J. Biol. Chem.263:15264-15269].

Eukaryotic translation initiation factor 5A (eIF-5A) is an essentialprotein factor approximately 17 kDa in size, which is involved in theinitiation of eukaryotic cellular protein synthesis. It is characterizedby the presence of hypusine [N-(4-amino-2-hydroxybutyl)lysine], a uniquemodified amino acid and known to be present only in eIF-5A. Hypusine isformed post-translationally via the transfer and hydroxylation of thebutylamine group from the polyamine, spermidine, to the side chain aminogroup of a specific lysine residue in eIF-5A. Activation of eIF-5Ainvolves transfer of the butylamine residue of spermidine to the lysineof eIF-5A, forming hypusine and activating eIF-5A. In eukaryotes,deoxyhypusine synthase (DHS) mediates the post-translational synthesisof hypusine in eIF-5A. The hypusine modification has been shown to beessential for eIF-5A activity in vitro using a methionyl-puromycinassay.

Hypusine is formed on eIF-5A post-translationally through the conversionof a conserved lysine residue by the action of deoxyhypusine synthase(DHS; EC 1.1.1.249) and deoxyhypusine hydroxylase (DOHH; EC 1.14.99.29).DHS cDNA has been directly sequenced or predicted from genomic sequencesin dozens of plant species, including Arabidopsis thaliana (GenBankAccession No. NM_120674), alfalfa (U.S. Pat. No. 8,563,285), banana(GenBank Accession No. XM_009405857), camelina (GenBank Accession No.XP_010452500), canola (GenBank Accession No. XM_013859772), carnation(GenBank Accession No. AF296080), cocoa (GenBank Accession No.CGD0006914), coffee (GenBank Accession No. GR986281), soybean (GenBankAccession No. BM092515), tobacco (GenBank Accession No. NM 001325620),tomato (GenBank Accession No. NM 001247566), wheat (GenBank AccessionNo. FJ376389), and many others. DOHH cDNA sequences have also beenidentified in some plants, including Medicago truncatula (GenBankAccession No. XM_013594404).

DHS converts a conserved lysine residue of eIF-5A to deoxyhypusinethrough the addition of a butylamine group derived from spermidine. Thisintermediate form of eIF-5A is then hydroxylated by DHH to becomehypusine [Park et al. (1997) Biol. Signals 6:115-123]. Both thedeoxyhypusine and the hypusine form of eIF-5A are able to bind cDNA invitro [Liu et al. (1997) Biol. Signals 6:166-174]. Although the functionof eIF-5A is not fully understood, there is some evidence that it mayregulate cell division [Park et al. (1998) J. Biol. Chem.263:15264-15269; Tome et al. (1997) Biol. Signals 6:150-156], andsenescence. [Wang et al. (2001) J. Biol. Chem. 276:17541-17549]. Itappears that several organisms are known to have more than one isoformof eIF-5A, which would suit the premise that each isoform is a specificshuttle to specific suites of mRNAs that are involved in such processesas cell division and senescence.

Wang et al. demonstrated that an increased level of DHS cDNA correlateswith fruit softening and natural and stress-induced leaf senescence oftomato [Wang et al. (2001) J. Biol. Chem. 276:17541-17549; (2003) PlantMolecular Biology 52: 1223-1235; and (2005) Plant Physiology138:1372-1382]. Furthermore, when the expression of DHS was suppressedin transgenic tomato plants by introducing a DHS antisense cDNA fragmentunder the regulation of a constitutive promoter, the tomato fruit fromthese transgenic plants exhibited dramatically delayed senescence asevidenced by delayed fruit softening and spoilage. See U.S. Pat. Nos.6,878,860, 6,900,368, 7,070,997, and 7,226,784. Since DHS is known toactivate eIF-5A, these data suggest that the hypusine-modified eIF-5A(active eIF-5A) may regulate senescence through selective translation ofmRNA species required for senescence. This is further demonstratedthrough the down-regulation of DHS in Arabidopsis thaliana (“AT”) byantisense of the full length or 3′UTR cDNA under the control of aconstitutive promoter. By down regulating Arabidopsis thaliana DHS(“AT-DHS”) expression and making it less available for eIF-5Aactivation, senescence was delayed by approximately 2 weeks [See Duguayet al. (2007) Journal of Plant Physiology 164:408-420 & U.S. Pat. No.7,226,784]. Not only was senescence delayed, but also an increase inseed yield, an increase in stress tolerance and an increase in biomasswere observed in the transgenic plants, where the extent of eachphenotype was determined by the extent of the down-regulation of DHS.

Although down-regulation of DHS in plants by means of anti sensetransgenic plants is expected to generate plants with advantageousagronomic properties, such as resistance to stress, delayed senescence,and increased yields, transgenic plants in general have severaldisadvantages. Creation of transgenic DHS plants requires theintroduction of foreign DNA, including the antisense gene, which oftenuse viral promoters for strong expression, as well as selection genes.Furthermore, viral promoters are often recognized by the plant andturned-off, leading to loss of antisense expression in futuregenerations of transgenic plants. A better strategy for down-regulationof DHS in plants, for instance alfalfa, is to use genome editing tomodify the gene in order reduce or eliminate the activity of thetranslated DHS protein. Tomato, Arabidopsis thaliana and many otherplants only have one copy of DHS in their genome, as shown by Southernblot [Wang et al. (2001) J. Biol. Chem. 276:17541-17549] and full genomesequencing. Since alfalfa is a tetraploid plant, using a genome editingtechnique could disrupt in separate progeny from the same experiment:one out of the four DHS copies found in its genome thereby reducing DHSactivity in the plant tissues by approximately 25%, two out of the fourDHS copies found in its genome thereby reducing activity byapproximately 50%, or three out of the four DHS copies found in itsgenome thereby reducing activity by approximately 75%. Screeningindependent progeny expressing each of these residual activity levelscould lead to identification of clones that demonstrate the maximumdegree of improved resistance to stress and delayed senescence viaincomplete hypusination of eIF-5A isoforms involved in stress andsenescence pathways. It is unlikely to find progeny that have all fourDHS copies disrupted since this should be a lethal event, given thathomozygous knockout of DHS has been demonstrated to be lethal in miceand yeast [Templin et al. (2011) Cell Cycle 10:1043-9; Sasaki et al.(1996) FEBS Lett. 384:151-4].

Genome editing makes use of various technologies to manipulate thegenome of either plants or animals by inserting, deleting, orsubstituting specific genetic sequences in a highly specific manner.Many genome editing methods exist, and include, but are not limited to,use of transgenic DNA sequences flanked by sequences homologous to theintended site of modification (homologous recombination), or methodsusing engineered nucleases, including Meganucleases, zinc fingernucleases, (ZFNs), CRISPR-Cas9, CRISPR-Cms1, transcriptionactivator-like effector-based nucleases (TALEN), and ARC nuclease(ARCUS). In all these systems, nucleases create site-specificdouble-stranded DNA breaks which are then repaired via homologousrecombination or nonhomologous end-joining to create the desiredmutation.

An example of homologous recombination is the rapid trait developmentsystem (RTDS) [Beetham et al. (1999) Proc. Natl. Acad. Sci. USA96:8774-8778; Kochevenko and Willmitzer (2003) Plant Physiol.132:174-184]. RTDS uses a Gene Repair Oligonucleotide (GRON) tointroduce a mismatch error into the sequence of a targeted gene in ahighly specific manner. This mismatch is then repaired by the plant'snatural DNA repair system that uses the GRON as a template in order tocreate the desired modification.

The clustered regularly interspaced short palindromic repeats(CRISPR)/Cas9 genome editing system has been used in a wide variety oforganisms, including monocot and dicot plants. One or more single guideRNA (sgRNA), with a target sequence homologous to the desired gene beingtargeted for editing, is introduced along with Cas9 nuclease, and areused to direct the Cas9 protein to a specific genomic site [reviewed byMa and Liu (2016) Curr. Protoc. Mol. Biol, DOI: 10.1002/cpmb.10].Similar experiments can be designed using other double strandednucleases, such as Cms1 [Begemann and Gray, U.S. Pat. No. 9,896,696].

Transcription activator-like effectors (TALE) are naturally occurringtranscription effectors that use a simple code of tandem repeats thatallows for customizable generation of TALEs that recognize a DNAsequence with high specificity. When combined with a functional domain,such as FokI nuclease (TALEN), targeted gene disruption is possible.Binding of the TALEN nuclease to its engineered recognition sequenceresults in double-stranded DNA break and subsequent recruitment of thenon-homologous end joining repair machinery, resulting in either smalldeletions or insertions that lead to disrupted gene function. TALEN hasbeen used to modify economically important food crops and biofuels andis being explored in correcting genetic errors that underpin certainhuman diseases. TALEN can also be combined with the simultaneousintroduction of a targeting segment of DNA that contains homology to thecleavage site in order to specifically introduce the targeted sequencesinto the locus using homologous recombination.

Meganucleases (also called homing endonucleases) are nucleases thatrecognize large sequences (12-40 base pairs) that occur very rarely, andideally only once, in the genome [Porteus et al. (2005) Nat. Biotechnol.23:967-73]. Meganucleases normally recognize palindromic sequences,however, by producing a pair of monomers which recognize two differenthalf-sites which, as heterodimers, form a meganuclease that cleaves anon-palindromic site. ARCUS is a genome editing technology that is basedon ARC nuclease, a totally synthetic homing endonuclease-like enzymethat is derived from a naturally occurring homing endonuclease. ARCnuclease can be customized to recognize a specific DNA sequence,allowing a precise DNA break, often at only a single site in the genome.This DNA break allows genome modification, including insertions,deletions, or substitutions, by homologous recombination.

The function of the DHS enzyme is well understood. The deoxyhypusinesynthase reaction catalyzed by DHS involves interaction with threedifferent substrates: spermidine, NAD, and eIF-5A precursor protein[eIF-5A(Lys)]. The first step of the reaction is the NAD-dependentdehydrogenation of spermidine, the second step involves trans-iminationto form the DHS-imine intermediate, the third step involvestrans-imination for the eIF-5A-imine intermediate, and the fourth stepis the enzyme-coupled reduction of the eIF-5A-imine intermediate [Joe etal. (1997) J. Biol. Chem. 272:32679-685]. The enzyme-imine intermediatebond is formed between the 4-amino-butyl moiety of spermidine and theε-amino group of Lys³²⁹ in the human enzyme [Joe et al. J. Biol. Chem.(1997) 272:32679-685]. Upon addition of the eIF-5A(Lys) precursor, thebutylamine group is transferred to Lys⁵⁰ of human eIF-5A and thenreduced to form deoxyhypusine. Lys³²⁹ of human DHS is therefore crucialfor DHS enzymatic activity, since it is absolutely required for thetransfer of the butylamine group from spermidine to eIF-5A.

Lys³²⁹ has been demonstrated to be a critical residue in the active siteof human DHS (Table 1). Single point mutations of this conserved lysine(K329A or K329R) completely disrupt the ability of human DHS to catalyzethe transfer of the butylamine group of spermidine to the conservedLys⁵⁰ residue of eIF5A to generate deoxyhypusine [Joe et al. J. Biol.Chem. (1997) 272:32679-685; Wolff et al. (1997) J. Biol. Chem.272:15865-71; Lee et al. Biochem J. (2001) 355:841-9]. The correspondingresidue in yeast, Lys³⁵⁰, has also been demonstrated to be critical fordeoxyhypusine synthase activity [Wolff and Park (1999) Yeast 15:43-50].The region surrounding and including Lys³²⁹ in human DHS is highlyconserved in plant species and a conserved region of seven amino acidsin the active site from Glu³²³ to Lys³²⁹ (human DHS numbering) isabsolutely conserved in examined plant DHS sequences (FIG. 1). This highdegree of conservation suggests that mutation of the correspondingresidue in plants will result in complete abolition of the ability ofthe expressed enzyme to catalyze the synthesis of deoxyhypusine.

TABLE 1 Active regions of Human DHS. DHS Function Residues ReferenceIntermediate formation Lys³²⁹ Wolff et al. (1997) with the butylamine J.Biol. Chem. moiety of spermidine 272: 15865-71 NAD binding Asn¹⁰⁶,Asp²³⁸, His²⁸⁸, Lee et al. (2001) Asp³¹³, Asp³⁴² Biochem. J. 355: 841-9Spermidine binding His²⁸⁸, Trp³²⁷, Lys³²⁹, Lee et al. (2001) andreaction Asp³¹⁶, Glu³²³ Biochem. J. 355: 841-9 Wolff et al. (1997) J.Biol. Chem. 272: 15865-71 Wolff et al. (2000) J. Biol. Chem. 275: 9170-7

Amino acid residues that have been identified as being involved in NAD⁺binding in human DHS, Asn¹⁰⁶, Asp²³⁸, His²⁸⁸, and Asp³¹³, are conservedin all examined plant DHS sequences (FIG. 1 and Table 1). Residues whichparticipate in spermidine binding and catalysis of deoxyhypusinesynthesis in human DHS, His²⁸⁸, Trp³²⁷, Lys³²⁹, Asp³¹⁶, and Glu³²³, arealso conserved in plant DHS sequences [FIG. 1 and Table 1; Lee et al.(2001) Biochem J. 355:841-9]. The critical residue Lys²⁸⁷, which isimportant for covalent intermediate formation [Joe et al. (1997) J.Biol. Chem. 272:32679-685] and Lys³²⁹, which is the catalytic center andcritical for the enzymatic activity of DHS and formation of intermediatewith the transfer of the butylamine moiety of spermidine are also highlyconserved (FIG. 1 and Table 1).

Although the sequences are highly conserved in the active regions ofDHS, the numbering of important residues differs in plant speciescompared to human. The numbering of important residues in M. sativa andthe corresponding residue No. of the human DHS is shown in Table 2. Forexample, Lys²⁸⁷ and Lys³²⁹ of human DHS correspond to Lys²⁹² and Lys³³⁴of M. sativa, respectively.

TABLE 2 Amino acid residues in human DHS demonstrated to be critical toDHS functional activity and the corresponding residues in alfalfa (M.sativa). Human DHS Residue M. sativa DHS Residue K329 K334 K287 K292K338 K343 W327 W332 K141 K144 D313 D318

Other residues involved in the binding of NAD⁺ and/or spermidine havealso been identified as being critical for human DHS enzyme activity(Table 3). These include Lys²⁸⁷, mutation of which leads to a 99%reduction (with respect to wild-type enzyme) in the ability of theenzyme to cleave spermidine and synthesize deoxyhypusine [Joe et al.(1997) J. Biol. Chem. 272:32679-685]. Lys²⁸⁷, a highly conservedresidue, participates in formation of a side pocket cavity that appearsto be important for functional activity [Lee et al. (2001) Biochem J.355:841-9; Umland et al. (2004) J. Biol. Chem. 279:28697-705]. Thisresidue is adjacent to His²⁸⁸, which is predicted to play a role in theNAD dehydrogenation of spermidine, perhaps by acting as a protonacceptor/donor [Umland et al. (2004) J. Biol. Chem. 279:28697-705].Mutation of His²⁸⁸ also resulted in a nearly complete loss of spermidinebinding and enzymatic activity [Lee, 2001]. Other residues involved inspermidine binding also resulted in a drastic reduction in deoxyhypusinesynthase activity when mutated to alanine. These include: D243A, W327A,H288A, D316A, E323A, K329A, and K329R (see Table 3; Lee et al. (2001)Biochem J. 355:841-9]. Along with Lys³²⁷, residues Asp³¹⁶, His²⁸⁸, andGlu³²³, appear to help define a side pocket cavity [Umland et al. (2004)J. Biol. Chem. 279:28697-705]. Residues involved in NAD⁺ binding werealso found to result in an almost complete loss of enzymatic activitywhen mutated to alanine. These include: D342A, D313A, D238A, E137A (seeTable 3; Lee et al. (2001) Biochem. J. 355:841-9].

Certain residues in human DHS have been identified, e.g., K¹⁴¹, thatwhen mutated result in a DHS protein which retains a certain measure ofactivity. For example, the K141R mutation in human DHS resulted inretention of 20% of the DHS activity (see Table 3; Joe et al. (1997) J.Biol. Chem. 272:32679-685]. Rather than creating a deletion, insertion,or substitution in DHS that completely abrogates deoxyhypusine synthaseactivity, it may be possible to introduce a specific single pointmutation in a functionally important residue, such as K⁴¹(K¹⁴⁴ in M.sativa, Table 2) that would reduce the activity of the modified gene butnot eliminate it. Screening independent progeny expressing a variety ofresidual activity levels, depending on whether 1, 2, 3 or 4 gene copieshave been modified, could lead to identification of clones thatdemonstrate the maximum degree of improved resistance to stress anddelayed senescence via incomplete hypusination of eIF-5A isoformsinvolved in stress and senescence pathways. In this case, it may bepossible to find progeny that have all four DHS copies modified sincethere may be enough residual DHS activity present to prevent lethality.

TABLE 3 Previously described mutations, substitutions or deletions inthe 369-aa Human DHS protein and their effects on DHS activities,including NAD⁺ binding, spermidine binding, eIF5A(Lys) binding, anddeoxyhypusine synthase activity. DHS Mutation Phenotype ReferenceN-terminal deletion of Complete loss of activity Joe et al. (1995) Met₁to Ala₄₈ J. Biol. Chem. 270: 22386-392 N-terminal deletion of Completeloss of activity Joe et al. (1995) deletion of Met₁ to Cys₉₇ J. Biol.Chem. 270: 22386-392 Internal deletion of Complete loss of activity Joeet al. (1995) Asp₂₆₂ to Ser₃₁₇ J. Biol. Chem. 270: 22386-392 Internaldeletion of amino Complete loss of activity Joe et al. (1995) acids 269to 317 J. Biol. Chem. 270: 22386-392 C-terminal deletion of Completeloss of activity Joe et al. (1995) Asp₃₃₃ to Asp₃₆₉ J. Biol. Chem. 270:22386-392 K329A Loss of intermediate formation with the Wolff et al.(1997) butylamine residue of spermidine; Complete J. Biol. Chem. loss ofdeoxyhypusine synthesis; ~6% of 272: 15865-71 spermidine cleavageactivity Joe et al. (1997) J. Biol. Chem. 272: 32679-685 K329R Completeloss of deoxyhypusine synthesis and Joe et al. (1997) spermidinecleavage J. Biol. Chem. 272: 32679-685 K287R or K287A <1% ofdeoxyhypusine synthesis activity and Joe et al. (1997) spermidinecleavage J. Biol. Chem. 272: 32679-685 K338R <10% of deoxyhypusinesynthase activity; Joe et al. (1997) 35% spermidine cleavage activity J.Biol. Chem. 272: 32679-685 K141R ~20% of deoxyhypusine synthesisactivity; Joe et al. (1997) Small reduction in spermidine cleavage J.Biol. Chem. 272: 32679-685 W327A ~2% spermidine cleavage activity; <<1%Wolff et al. (2000) NADH forming activity J. Biol. Chem. 275: 9170-7D313A Significantly reduced eIF5A(Lys) binding and Lee et al. (2001) NADbinding Biochem. J. 355: 841-9 D243A; W327A; H288A; Almost complete lossof spermidine binding Lee et al. (2001) D316A; E323A; K329A; and enzymeactivity Biochem. J. or K329R 355: 841-9 D342A; D313A; D238A; Almostcomplete loss of NAD binding and Lee et al. (2001) orE137 enzymeactivity Biochem. J. 355: 841-9

The ability to construct homozygous mutations that reduce but don'teliminate catalytic function of the DHS protein is particularlyimportant in crops grown from hybrid seeds. Examples include corn,wheat, soybeans, grain sorghum, cotton, peanuts and many other crops. Inthese cases, elite inbred strains are used as parents that arehomozygous at most loci. One or both parental lines having reducedactivity due to targeted mutations in their DHS genes would beparticularly advantageous in the resulting hybrid seeds sold to farmers.

Presently, there is no widely applicable method for controlling theonset of programmed cell death (including senescence) caused by eitherinternal or external, e.g., environmental stress, factors. It is,therefore, of interest to develop senescence modulating technologiesthat are applicable to all types of plants and that are effective at theearliest stages in the cascade of events leading to senescence. Genomeediting of DHS is a possible solution to reduce loss in plant yields dueto environmental stress, as well as increase shelf life of perishableproduce such as fruits, vegetables and flowers.

SUMMARY OF THE INVENTION

The present invention provides protein sequences of deoxyhypusinesynthase from plant species, including alfalfa, and the polynucleotidesthat encode these proteins, including genomic sequences. The presentinvention also relates to methods involving genome editing or baseediting (involving either deletions, insertions, or substitutions) todisrupt the activity of these DHS proteins by targeting amino acidresidues critical for DHS deoxyhypusine synthase enzymatic activity.

The present invention provides a method for genetic modification ofplants to control the onset of senescence, either age-related senescenceor environmental stress-induced senescence. One of several genomeediting technologies, including but not limited to RTDS, TALEN,CRISPR-Cas9, CRISPR-Cms1, ARCUS or base editing, is used to introduce adeletion, insertion, or substitution in the region of an amino acidresidue critical for DHS function, in order to lead to the reduction orelimination of the DHS protein activity, thereby reducing the level offunctionally active endogenous senescence-induced DHS protein, andreducing and/or preventing activation of eIF-5A and ensuing downstreamexpression of the genes that mediate senescence.

Methods and compositions are provided herein for the control of DHSprotein activity involving sequence targeting, such as genomeperturbation or gene-editing, that relate to the CRISPR-Cms1 system andcomponents thereof as set forth in U.S. Pat. No. 9,896,696; Begemann etal. (2017) Scientific Reports 7, Article 11606; and Begemann et al.(2017) bioRxiv (DOI: Cms1 was previously referred to as Csm1 (see U.S.Pat. No. 9,896,696).

The disclosures of which are incorporated herein by reference. Incertain embodiments, the Type V CRISPR enzyme is a Cms enzyme, e.g., aCms1 ortholog, particularly MiCms1. The methods and compositions includenucleic acids to bind target DNA sequences. This is advantageous asnucleic acids are much easier and less expensive to produce than, forexample, peptides, and the specificity can be varied according to thelength of the stretch where homology is sought. Complex 3-D positioningof multiple fingers, for example is not required. The Cms1 enzyme isalso smaller than Cas9, works more efficiently in plants, and leavessticky ends instead of blunt ends after DNA cutting.

An alternative to genome editing for making useful changes to the DHSgenomic sequence is CRISR base editing, particularly to make pointmutations in the coding region of DHS genes at the active site or any ofits NAD⁺ binding sites. This recent technology uses catalyticallyinactivated CRISPR nucleases, such as dead Cas9 (dCas9), fused todiverse functional domains for targeting genetic modifications tospecific DNA sequences [Eid et al. Biochem J. (2018) 475: 1955-1964]. Nodouble-strand breaks are generated, and the dCas9 targets adeninine orcytidine deaminases to convert their target nucleotides into other DNAbases. Many examples of specific targets of base editing the Lys₁₄₉codon (one of the NAD+ binding sites) in 29 plant species are providedin Example 13.

Using the methods of the invention, genome edited or base edited plantsare generated and monitored for growth, development and either naturalor delayed senescence. Plants or detached parts of plants (e.g.,cuttings, flowers, vegetables, fruits, seeds or leaves) exhibitingprolonged life or shelf life, (e.g., extended life of flowers, reducedfruit or vegetable spoilage), enhanced biomass, increased seed yield,increased resistance to physiological disease (e.g., blossom end rot,reduced seed aging and/or reduced yellowing of leaves) due to reductionin the level of senescence-induced DHS, senescence-induced eIF-5A orboth are selected as desired products having improved propertiesincluding reduced leaf yellowing, reduced petal abscission, reducedfruit and vegetable spoilage during shipping and storage. These superiorplants are propagated. Similarly, plants exhibiting increased resistanceto environmental stress (e.g., high or low temperatures, drought, lownutrient levels, high salt, crowding, pathogen infection, and/orphysiological disease) are selected as superior products.

The type of plant which can be used in the methods of the invention isnot limited and includes, for example, ethylene-sensitive andethylene-insensitive plants; fruit bearing plants such as apricots,apples, oranges, bananas, grapefruit, pears, tomatoes, strawberries,avocados, grapes, etc. In some embodiments, the plant is a vegetablesuch as carrots, peas, lettuce, cabbage, turnips, potatoes, broccoli,asparagus, etc. In some embodiments, the plant is a flower such ascarnations, roses, mums, etc. In some embodiments, the plant is anagronomic crop plant and includes forest species such as corn, rice,soybean, alfalfa, wheat, cotton, sugarbeet, canola, sorghum, sunflower,Camelina, peanuts, trees and the like. In general, any plant that cantake up DNA molecules for genome editing can be used in the methods ofthe invention and may include plants of a variety of ploidy levels,including haploid, diploid, tetraploid and polyploid. The plant may beeither a monocotyledon or dicotyledon.

BRIEF DESCRIPTION OF THE DRAWINGS

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FIG. 1. Alignment of various plant DHS proteins with the human DHSprotein. Lane 1: Homo sapiens DHS (Accession No. NP 001921) (SEQ ID NO:10); Lane 2: Arabidopsis thaliana (Accession No. NP 196211) (SEQ ID NO:11); Lane 3: Camelina sativa DHS (accession number XP 010452500) (SEQ IDNO: 44); Lane 4: Brassica rapa DHS (accession number XP 009122196) (SEQID NO: 36); Lane 5: Brassica napus DHS (accession number XP_013715226)(SEQ ID NO: 38); Lane 6: Gossypium hirsutum DHS (accession number XP016700362) (SEQ ID NO: 32); Lane 7: Populus deltoides DHS (sequence isnot in GenBank; sequence is from FIGS. 109b in US Patent Application No.US 2010/0333233) (SEQ ID NO: 46); lane 8: Camellia sinensis DHS(www.plantkingdomgdb.com/tea tree/data/pep/Teatree Protein.fas; Xia(2017) Molecular Plant 10:866-877) (SEQ ID NO: 54); lane 9: Vitisvinifera DHS (SEQ ID NO: 52); Lane 10: Medicago sativa DHS (sequence isnot in GenBank; sequence is from FIGS. 107a and 107b in U.S. Pat. No.8,563,285) (SEQ ID NO: 2); Lane 11: Cicer arietinum DHS (accessionnumber XP 004504559.1) (SEQ ID NO: 50); Lane 12: Arachis duranensis DHS(accession number XP 015966414) (SEQ ID NO: 18); Lane 13: Arachisipaensis DHS (accession number XP_016203820) (SEQ ID NO: 20); Lane 14:Glycine max DHS 1 (accession number NP 001235604) (SEQ ID NO: 22); Lane15: Glycine max DHS 2 (accession number NP 001237752) (SEQ ID NO: 24);Lane 16: Phaseolus vulgaris DHS (accession number XP 007152935) (SEQ IDNO: 48); Lane 17: Beta vulgaris subsp. vulgaris (accession number XP010681287) (SEQ ID NO: 34); Lane 18: Solanum lycopersicum DHS (accessionnumber NP 001234495) (SEQ ID NO: 12); Lane 19: Solanum tuberosum DHS(accession number XP 006348136) (SEQ ID NO: 42); lane 20: Coffeacanephora DHS (SEQ ID NO: 56); Lane 21: Triticum aestivum DHS (accessionnumber ACP28133) (SEQ ID NO: 13); Lane 22: Oryza sativa Japonica GroupDHS (accession number XP 015628158) (SEQ ID NO: 30); Lane 23: Zea maysDHS 1 (accession number NP 001149084) (SEQ ID NO: 26); Lane 24: Sorghumbicolor DHS (accession number XP 002466487) (SEQ ID NO: 40); Lane 25:Zea mays DHS 2 (accession number NP 001130806) (SEQ ID NO: 28); Lane 26:Musa acuminate DHS (accession number XP 009404132) (SEQ ID NO: 14); lane27: Theobroma cacao DHS (www.cacaogenomedb.org CGD0006914) (SEQ ID NO:53); lane 28: Partial amino acid sequence of Mentha longifolia DHS (MintGenomics Resource; www.langelabtools.wsu.edu/mgr/; TRINITY DN66685clg8i1) (SEQ ID NO: 55); lane 29: Lactuca sativa DHS (accession numberAAU34016) (SEQ ID NO: 64). Consensus sequence (SEQ ID NO: 51) is shownin the last lane (! is either of L, Q, I or V, $ is either of L or M, %is either of R, F or Y, # is any of H, T, K, N, A, D, Q or E). Aconserved region of seven amino acids in the active site from E323 toK329 (human DHS numbering) is bolded black and enclosed with a box. NAD⁺binding regions are bolded in purple. The catalytic lysine residue (K329of human DHS) is bolded black and underlined. Other important residuesare highlighted (human DHS numbering): blue (K287), green (K338), yellow(K141), red (W327), purple (D313).

FIG. 2. Phylogenetic analyses of DHS proteins from different organismsrevealing that DHS proteins are closely related. The dendrogram showsthe evolutionary relationship between twenty-eight different DHSproteins from twenty-six different organisms. The phylogenetic treeclustering was conducted on all the proteins listed in FIG. 1 usingBioEdit 7.2 software [bioedit.software.informer.com/7.2/] with ClustalWsequence alignment and the Unweighted Pair Group Method with ArithmeticMean (UPGMA) phylogenetic tree generating algorithm.

FIG. 3. Genomic DNA of DHS genes from various plant species. Boxesrepresent exons. Lines represent introns. The coding sequence is shaded.Red lines and down arrows (↓) represent the lysine residue that forms acovalent intermediate with a butylamine moiety. Pink lines and asterisks(*) represent the active site.

FIG. 4. Nucleotide sequence of the DHS gene of B. napus [SEQ ID NO: 37].Start and stop codons at 84 and 1188, respectively, are italicized andunderlined. Regions targeted by the three CRISPR guide RNAs are in boldand underlined starting at 600, 635, and 1157. The guanine (G)nucleotide deleted in the DHS gene of B. napus line #16 at 569 and theresulting TGA stop codon at 652 are highlighted and underlined.

FIG. 5. B. napus line #16 plant (left) and a wild-type control B. napusplant (right). B. napus line 16 is a dwarf with darker green leaves,delayed flower emergence, and delayed senescence.

FIG. 6. Alignment of DNA sequences from GE0568-DHS1 rice callusesdisplaying the locations of internal deletions. Bold text represents theinverse complement of the MiCms1 PAM site (TTTC).

FIG. 7. Alignment of DNA sequences from GE0568-DHS1 T0 rice plantsdisplaying the locations of internal deletions and inserts. Bold textrepresents the inverse complement of the MiCms1 PAM site (TTTC).

FIG. 8. Molecular analysis of T0 plants arising from callus 30. (A) PCRamplification of DHS1 from plant extracts (left). T7 nuclease treatmentof the PCR products (right). The appearance of a lower band aftertreatment with T7 nuclease indicates heteroduplex formation and suggeststhe presence of an internal deletion. (B) Sequence alignments of the twoDHS1 alleles isolated from T0 plants regenerated from callus 30T andcallus 30S. Red text represents the inverse complement of the MiCms1 PAMsite (TTTC).

FIG. 9. Strategy for disrupting the active site of rice DHS1. (A)Genomic structure of the rice DHS1 gene with introns as thin crookedlines, and exons as filled black boxes. The active site (EAVSWGK SEQ IDNO: 546) is in Exon 6. (B) Sequence of WGK and 6 downstream codons,including the PAM site and gRNA sequence. The guide RNA is designed totarget the sense strand (CDS) and is designed based on the antisensesequence.

FIG. 10. Sequence alignment of the two DHS1 alleles isolated from theheterozygous T0 line #2. The lower sequence has a 10 bp internaldeletion resulting in the formation of a TGA stop codon (bold) thattruncates the C-terminal 41 amino acids. The bold text in the antisensestrand represents a PAM site.

FIG. 11. Visual appearance of T1 plants with two wild-type DHS1 alleles(left) or one truncated DHS1 allele and one wild-type allele (right).The heterozygote has a darker green coloration.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, a “corresponding residue” refers to any amino acid in aDHS protein, that upon alignment with a second DHS protein amino acidsequence (e.g., human DHS), which is in a different location based onnumbering from the N-terminus to C-terminus, and would be in the samelocation but for the different numbering due to gaps introduced by anysequence alignment. Examples of corresponding residues are describedherein and, for example, in FIG. 1. Examples of plant DHS amino acidsequences and corresponding nucleotide sequences include, but are notlimited to, the sequence of the coding regions of Medicago sativa DHS(SEQ ID NO: 1) and its corresponding amino acid translation (SEQ ID NO:2); cDNA sequence of Arabidopsis thaliana DHS (GenBank Accession No.NM_120674) (SEQ ID NO: 4); amino acid sequence of Arabidopsis thalianaDHS (GenBank Accession No. NP_196211) (SEQ ID NO: 11); cDNA sequence ofSolanum lycopersicum DHS (GenBank Accession No. NM_001247566) (SEQ IDNO: 5); cDNA sequence of Triticum aestivum DHS (GenBank Accession No.FJ376389) (SEQ ID NO: 6); cDNA sequence of Musa acuminate DHS (GenBankAccession No. XM_009405857) (SEQ ID NO: 7); partial cDNA sequence ofMedicago truncatula DHS (GenBank Accession No. XM_013591001) (SEQ ID NO:8); cDNA sequence of Medicago sativa DHS (previously described in U.S.Pat. No. 8,563,285) (SEQ ID NO: 9); amino acid sequence of Solanumlycopersicum DHS (GenBank Accession No. NP_001234495) (SEQ ID NO: 12);amino acid sequence of Triticum aestivum DHS (GenBank Accession No.ACP28133) (SEQ ID NO: 13); amino acid sequence of Musa acuminate DHS(GenBank Accession No. XP_009404132) (SEQ ID NO: 14); partial amino acidsequence of Medicago truncatula DHS (GenBank Accession No. XP_013446455)(SEQ ID NO: 15); amino acid sequence of Medicago sativa DHS (previouslydescribed in U.S. Pat. No. 8,563,285) (SEQ ID NO: 2); cDNA sequence ofArachis duranensis DHS (GenBank Accession No. XM_016110928) (SEQ ID NO:17); amino acid sequence of Arachis duranensis (GenBank Accession No.XP_015966414) (SEQ ID NO: 18); cDNA sequence of Arachis ipaensis DHS(GenBank Accession No. XM_016348334) (SEQ ID NO: 19); amino acidsequence of Arachis ipaensis (GenBank Accession No. XP_016203820) (SEQID NO: 20); cDNA sequence 1 of Glycine max DHS (LOC100305453) (GenBankAccession No. NM_001248675) (SEQ ID NO: 21); amino acid sequence 1 ofGlycine max (GenBank Accession No. NP 001235604) (SEQ ID NO: 22); cDNAsequence 2 of Glycine max DHS (LOC100499650) (GenBank Accession No. NM001250823) (SEQ ID NO: 23); amino acid sequence 2 of Glycine max(GenBank Accession No. NP_001237752) (SEQ ID NO: 24); cDNA sequence 1 ofZea mays DHS (GenBank Accession No. NM_001155612) (SEQ ID NO: 25); aminoacid sequence 1 of Zea mays (GenBank Accession No. NP_001149084) (SEQ IDNO: 26); cDNA sequence 2 of Zea mays DHS (GenBank Accession No.NM_001137334) (SEQ ID NO: 27); amino acid sequence 2 of Zea mays(GenBank Accession No. NP_001130806) (SEQ ID NO: 28); cDNA sequence ofOryza sativa Japonica Group DHS (GenBank Accession No. XM_015772672)(SEQ ID NO: 29); amino acid sequence of Oryza sativa Japonica Group(GenBank Accession No. XP_015628158) (SEQ ID NO: 30); cDNA sequence ofGossypium hirsutum DHS (LOC107915737) (GenBank Accession No.XM_016844873) (SEQ ID NO: 31); amino acid sequence of Gossypium hirsutum(GenBank Accession No. XP 016700362) (SEQ ID NO: 32); cDNA sequence ofBeta vulgaris subsp. vulgaris DHS (LOC104896269) (GenBank Accession No.XM 010682985) (SEQ ID NO: 33); amino acid sequence of Beta vulgarissubsp. vulgaris (GenBank Accession No. XP 010681287) (SEQ ID NO: 34);cDNA sequence of Brassica rapa DHS (LOC103846938) (GenBank Accession No.XM_009123948) (SEQ ID NO: 35); amino acid sequence of Brassica rapa(GenBank Accession No. XP_009122196) (SEQ ID NO: 36); cDNA sequence ofBrassica napus DHS (LOC106419026) (GenBank Accession No. XM_013859772)(SEQ ID NO: 37); amino acid sequence of Brassica napus (GenBankAccession No. XP 013715226) (SEQ ID NO: 38); cDNA sequence of Sorghumbicolor DHS (GenBank Accession No. XM_002466442) (SEQ ID NO: 39); aminoacid sequence of Sorghum bicolor (GenBank Accession No. XP 002466487)(SEQ ID NO: 40); cDNA sequence of Solanum tuberosum DHS (LOC102602600)(GenBank Accession No. XM_006348074) (SEQ ID NO: 41); amino acidsequence of Solanum tuberosum (GenBank Accession No. XP 006348136) (SEQID NO: 42); cDNA sequence of Camelina sativa DHS (LOC104734595) (GenBankAccession No. XM_010454198) (SEQ ID NO: 43); amino acid sequence ofCamelina sativa (GenBank Accession No. XP_010452500) (SEQ ID NO: 44);cDNA sequence of Populus deltoides DHS (previously described in U.S.Publication No. 2010/0333233) (SEQ ID NO: 45); amino acid sequence ofPopulus deltoides (previously described in U.S. Publication No.2010/0333233) (SEQ ID NO: 46); cDNA sequence of Phaseolus vulgaris DHS(GenBank Accession No. XM_007152873) (SEQ ID NO: 47); amino acidsequence of Phaseolus vulgaris (GenBank Accession No. XP_007152935) (SEQID NO: 48); cDNA sequence of Cicer arietinum DHS (LOC101505901) (GenBankAccession No. XM_004504502) (SEQ ID NO: 49); and amino acid sequence ofCicer arietinum (GenBank Accession No. XP 004504559) (SEQ ID NO: 50); acDNA sequence of Vitis vinifera (grape) DHS (ENAICBI156001CBI15600.3)(SEQ ID NO: 58); an amino acid sequence of Vitis vinifera (grape) DHS(SEQ ID NO: 52); a cDNA sequence of Theobroma cacao DHS(www.cacaogenomedb.org CGD0006914) (SEQ ID NO: 59); an amino acidsequence of Theobroma cacao DHS (www.cacaogenomedb.org CGD0006914) (SEQID NO: 53); a cDNA sequence of Camellia sinensis DHS (SEQ ID NO: 60); anamino acid sequence of Camellia sinensis DHS(www.plantkingdomgdb.com/tea tree/data/pep/Teatree Protein.fas; Xia etal. (2017) Molecular Plant 10:866-877) (SEQ ID NO: 54); a partial cDNAsequence of Mentha longifolia DHS (SEQ ID NO: 61); an amino acidsequence of Mentha longifolia DHS (Mint Genomics Resource;www.langelabtools.wsu.edu/mgd; TRINITY DN66685 clg8i1) (SEQ ID NO: 55);a partial cDNA sequence of Coffea canephora DHS (SEQ ID NO: 62); anamino acid sequence of Coffea canephora DHS (SEQ ID NO: 56); cDNAsequence of algal Guillardia theta CCMP2712 DHS (NCBI ReferenceSequence: XM 005831275.1) (SEQ ID NO: 63); an amino acid sequence ofalgal Guillardia theta CCMP2712 DHS (NCBI Reference Sequence:XP_005831332.1) (SEQ ID NO: 57); a cDNA sequence of Lactuca sativa(GenBank Accession No. AY731231) (SEQ ID NO: 65; sequence includingflanking regions); and an amino acid sequence of Lactuca sativa (SEQ IDNO: 64).

For purposes of clarification, the cultivated peanut species (Arachishypogaea) arose from a hybrid between two wild species of peanut: A.duranensis and A. ipaensis [Seijo et al. (2007) Am. J. Bot. 94(12)1963-71; Kochert et al. (1996) Am. J. Bot. 83:1282-91; Moretzsohn etal. (2013) Ann. Bot. 111:113-126.] The amino acid sequences of the DHSprotein between these parental diploids, A. duranensis and A. ipaensis,are identical (SEQ ID NOS: 18 and 20). Thus, the amino acid sequences ofthe DHS protein of the cultivated peanut (Arachis hypogaea) is expectedto be identical to both parents.

By genome editing endogenous DHS genes to delete or modify specificallydefined functionally critical residues, the resulting genome editedplants have no or substantially less DHS protein to activate eIF-5A. Asdiscussed earlier, eIF-5A must be activated by DHS to render itbiologically useful. Thus, by inhibiting or reducing the activity of DHSby genome editing, the resulting genome edited plants will have reducedactive eIF-5A. These genome-edited plants will exhibit an increase inbiomass of the plant, increased seed yield and/or increased seed size,and also be expected to be more tolerant to abiotic stresses and, in thecase of plants producing perishable fruits or vegetables, extendedpost-harvest shelf life.

Further evidence to support the contention that DHS and eIF-5A playregulatory roles in senescence was provided by treating carnationflowers with inhibitors that are specific for DHS. Spermidine and eIF-5Aare the substrates of DHS reaction [Park et al. (1993) Biofactors4:95-104; Park et al. (1997) Biol. Signals. 6:115-123]. Several mono-,di-, and polyamines that have structural features similar to spermidineinhibit DHS activity in vitro [Jakus et al. (1993) J. Biol. Chem.268:13151-13159]. Some polyamines, such as spermidine, putrescine, andspermine, have been generally used to extend carnation vase life [Wangand Baker (1980) Hort. Sci. 15:805-806]. Flower petal senescence wasdelayed 6 days after harvest of carnations that were vacuum infiltratedwith a transient infection system expressing antisense DHS compared tountreated flowers [Hopkins et al. (2007) New Phytol. 175:201-214].

A further major loss in agriculture besides the loss of growth due tostress is post-harvest stress-induced senescence [McCabe et al. (2001)Plant Physiol. 127:505-516]. This is especially true for plants that arepartially processed, such as cut lettuce. A symptom of cutting lettuceis browning which is a result of phenolics production [Matile et al.(1999) Annu. Rev. Plant Physiol. Mol. Biol. 50:67-95]. A field trial oflettuce with antisense polynucleotides of lettuce eIF-5A (LeIF-5A) orantisense full length DHS demonstrated that the transgenic lettuce wassignificantly more resistant to browning after cutting than the controllettuce. It appears that even though stress induced senescence due toharvesting has distinct circuitry [Page et al. (2001) Plant Physiol.125:718-727], the translational control upstream of browning and likelyother senescence symptoms is regulated at least in part by DHS andeIF-5A. Downstream of the regulation of senescence are the executiongenes. These are the effectors of senescence and cause the metabolicchanges that bring on the senescence syndrome. It appears that eIF-5Aand DHS when down-regulated or reduced in activity are capable ofdampening down a whole range of symptoms caused by senescence.

EXAMPLES Example 1

Genomic DNA sequences were identified for 31 DHS genes from 28 plantspecies and human, and delineated into exons and introns. Two DHS genesare shown for Glycine max (soy), Rosa chinensis (rose), and Zea mays(maize). Other species (e.g., Solanum lycopersicum—tomato) may have morethan one DHS gene, even if only one is provided. Guide RNAs for editingDHS genes can be targeted to exons or introns within the genomic DNA.The exon-intron boundaries of these genomic DNAs are illustrated in FIG.3.

A genomic DNA sequence for a DHS gene of Arachis duranensis is SEQ IDNO: 66, and the corresponding exons and introns are SEQ ID NO: 67 to SEQID NO: 81. A genomic DNA sequence for a DHS gene of Arachis ipaensis isSEQ ID NO: 82, and the corresponding exons and introns are SEQ ID NO: 83to SEQ ID NO: 97. A genomic DNA sequence for a DHS gene of Arabidopsisthaliana is SEQ ID NO: 98, and the corresponding exons and introns areSEQ ID NO: 99 to SEQ ID NO: 111. A genomic DNA sequence for a DHS geneof Brassica napus is SEQ ID NO: 112, and the corresponding exons andintrons are SEQ ID NO: 113 to SEQ ID NO: 125. A genomic DNA sequence fora DHS gene of Brassica rapa is SEQ ID NO: 126, and the correspondingexons and introns are SEQ ID NO: 127 to SEQ ID NO: 139. A genomic DNAsequence for a DHS gene of Beta vulgaris subsp. Vulgaris is SEQ ID NO:140, and the corresponding exons and introns are SEQ ID NO: 141 to SEQID NO: 153. A genomic DNA sequence for a DHS gene of Cicer arietinum isSEQ ID NO: 154, and the corresponding exons and introns are SEQ ID NO:155 to SEQ ID NO: 167. A genomic DNA sequence for a DHS gene of Coffeacanephora is SEQ ID NO: 168, and the corresponding exons and introns areSEQ ID NO: 169 to SEQ ID NO: 181. A genomic DNA sequence for a DHS geneof Camelina sativa is SEQ ID NO: 182, and the corresponding exons andintrons are SEQ ID NO: 183 to SEQ ID NO: 195. A genomic DNA sequence fora DHS gene of Camellia sinensis is SEQ ID NO: 196, and the correspondingexons and introns are SEQ ID NO: 197 to SEQ ID NO: 209. A genomic DNAsequence for a DHS gene of Gossypium hirsutum is SEQ ID NO: 210, and thecorresponding exons and introns are SEQ ID NO: 211 to SEQ ID NO: 225. Agenomic DNA sequence for a DHS gene of Glycine max is SEQ ID NO: 226,and the corresponding exons and introns are SEQ ID NO: 227 to SEQ ID NO:240. A second genomic DNA sequence for a DHS gene of Glycine max is SEQID NO: 241, and the corresponding exons and introns are SEQ ID NO: 242to SEQ ID NO: 253. A genomic DNA sequence for a DHS gene of Homo sapiensis SEQ ID NO: 254, and the corresponding exons and introns are SEQ IDNO: 255 to SEQ ID NO: 271. A genomic DNA sequence for a DHS gene ofLactuca sativa is SEQ ID NO: 272, and the corresponding exons andintrons are SEQ ID NO: 273 to SEQ ID NO: 285. A genomic DNA sequence fora DHS gene of Musa acuminate is SEQ ID NO: 286, and the correspondingexons and introns are SEQ ID NO: 287 to SEQ ID NO: 299. A genomic DNAsequence for a DHS gene of Manihot esculenta is SEQ ID NO: 300, and thecorresponding exons and introns are SEQ ID NO: 301 to SEQ ID NO: 315. Agenomic DNA sequence for a DHS gene of Medicago sativa is SEQ ID NO:316, and the corresponding exons and introns are SEQ ID NO: 317 to SEQID NO: 331. A genomic DNA sequence for a DHS gene of Oryza sativaJaponica is SEQ ID NO: 332, and the corresponding exons and introns areSEQ ID NO: 333 to SEQ ID NO: 345. A genomic DNA sequence for a DHS geneof Populus deltoides is SEQ ID NO: 346, and the corresponding exons andintrons are SEQ ID NO: 347 to SEQ ID NO: 359. A genomic DNA sequence fora DHS gene of Phalaenopsis equestris is SEQ ID NO: 360, and thecorresponding exons and introns are SEQ ID NO: 361 to SEQ ID NO: 373. Agenomic DNA sequence for a DHS gene of Phaseolus vulgaris is SEQ ID NO:374, and the corresponding exons and introns are SEQ ID NO: 375 to SEQID NO: 387. A genomic DNA sequence for a DHS gene of Rosa chinensis isSEQ ID NO: 388, and the corresponding exons and introns are SEQ ID NO:389 to SEQ ID NO: 401. A second genomic DNA sequence for a DHS gene ofRosa chinensis is SEQ ID NO: 402, and the corresponding exons andintrons are SEQ ID NO: 403 to SEQ ID NO: 415. A genomic DNA sequence fora DHS gene of Sorghum bicolor is SEQ ID NO: 416, and the correspondingexons and introns are SEQ ID NO: 417 to SEQ ID NO: 429. A genomic DNAsequence for a DHS gene of Solanum lycopersicum is SEQ ID NO: 430, andthe corresponding exons and introns are SEQ ID NO: 431 to SEQ ID NO:443. A genomic DNA sequence for a DHS gene of Solanum tuberosum is SEQID NO: 444, and the corresponding exons and introns are SEQ ID NO: 445to SEQ ID NO: 457. A genomic DNA sequence for a DHS gene of Triticumaestivum is SEQ ID NO: 458, and the corresponding exons and introns areSEQ ID NO: 459 to SEQ ID NO: 472. A genomic DNA sequence for a DHS geneof Theobroma cacao is SEQ ID NO: 473, and the corresponding exons andintrons are SEQ ID NO: 474 to SEQ ID NO: 484. A genomic DNA sequence fora DHS gene of Vitis vinifera is SEQ ID NO: 485, and the correspondingexons and introns are SEQ ID NO: 486 to SEQ ID NO: 498. A genomic DNAsequence for a DHS gene of Zea mays is SEQ ID NO: 499, and thecorresponding exons and introns are SEQ ID NO: 500 to SEQ ID NO: 514. Asecond genomic DNA sequence for a DHS gene of Zea mays is SEQ ID NO:515, and the corresponding exons and introns are SEQ ID NO: 516 to SEQID NO: 531.

Example 2

An engineered homing endonuclease known as ARCUS is engineered toproduce a nuclease that would be capable of creating a double-strandedcleavage in a region of M. sativa DHS that is required for itsdeoxyhypusine synthase activity. These regions targeted for cleavagecould include the nucleic acids surrounding the regions that, whentranslated, include, for example either Lys³³⁴ (corresponds to Lys³²⁹ ofhuman DHS—see Table 3) or Lys²⁹² (Lys²⁸⁷ in human DHS). The DNA cleavagecreated by the engineered ARC nuclease would allow the creation of asmall deletion, insertion or single-base pair substitution in a targetedregion of plant DHS that is known to be critical for its deoxyhypusinesynthase activity. In M. sativa, the activity of the enzyme can bedisrupted by deleting one or more of the following residues: Lys³³⁴(corresponds to Lys³²⁹ of human DHS—see Table 3) or Lys²⁹² (Lys²⁸⁷ inhuman DHS). In one embodiment, the nucleotide region encoding the regionsurrounding, and including, Lys³³⁴ of M. sativa DHS would be deleted inorder to abolish deoxyhypusine synthase activity. In another embodiment,the nucleotide region encoding the region surrounding, and including,Lys²⁹² of M. sativa DHS would be deleted in order to abolishdeoxyhypusine synthase activity.

Example 3

An engineered transcription activator-like effector (TALE) combined witha functional domain, for example, a FokI nuclease (TALEN), is engineeredto produce a nuclease that would be capable of creating adouble-stranded cleavage in a region of M. sativa DHS that is requiredfor its deoxyhypusine synthase activity. These regions targeted forcleavage include the nucleic acids surrounding the region that, whentranslated, includes, for example either Lys³³⁴ (corresponds to Lys³²⁹of human DHS—see Table 3) or Lys²⁹² (Lys²⁸⁷ in human DHS). The DNAcleavage created by the engineered nuclease allows for a small deletion,insertion or single-base pair substitution in a targeted region of plantDHS that is known to be critical for its deoxyhypusine synthaseactivity. In M. sativa, the activity of the enzyme is disrupted bydeleting one or more of the following residues: Lys³³⁴ (corresponds toLys³²⁹ of human DHS—see Table 3) or Lys²⁹² (Lys²⁸⁷ in human DHS). Forexample, the nucleotide region encoding the region surrounding, andincluding, Lys³³⁴ of M. sativa DHS is deleted to abolish deoxyhypusinesynthase activity. In another example, the nucleotide region encodingthe region surrounding, and including, Lys²⁹² of M. sativa DHS isdeleted to abolish deoxyhypusine synthase activity.

Example 4

A sgRNA is engineered to produce a guide RNA capable of creating adouble-stranded cleavage in a region of M. sativa DHS that is requiredfor its deoxyhypusine synthase activity, when introduced into the plantalong with Cas9 (CRISPR-Cas9 system). These regions targeted forcleavage include the nucleic acids surrounding the region that, whentranslated, include, for example, either Lys³³⁴ (corresponds to Lys³²⁹of human DHS—see Table 3) or Lys²⁹² (Lys²⁸⁷ in human DHS). The DNAcleavage by cas9 allows for the creation of a small deletion, insertionor single-base pair substitution in a targeted region of plant DHS thatis known to be critical for its deoxyhypusine synthase activity. In M.sativa, the activity of the enzyme is disrupted by deleting one or moreof the following residues: Lys³³⁴ (corresponds to Lys³²⁹ of humanDHS—see Table 3) or Lys²⁹² (Lys²⁸⁷ in human DHS). For example, thenucleotide region encoding the region surrounding, and including, Lys³³⁴of M. sativa DHS is deleted in order to abolish deoxyhypusine synthaseactivity. In another example, the nucleotide region encoding the regionsurrounding, and including, Lys²⁹² of M. sativa DHS is deleted in orderto abolish deoxyhypusine synthase activity.

Example 5

A GRON is engineered to produce an oligonucleotide that creates amismatch error and subsequent repair using the GRON as a template(RTDS™), so as to create a desired substitution or deletion in a regionof M. sativa DHS that is required for its deoxyhypusine synthaseactivity. These regions targeted for cleavage include the nucleic acidssurrounding the region that, when translated, includes, for exampleeither Lys³³⁴ (corresponds to Lys³²⁹ of human DHS—see Table 3) or Lys²⁹²(Lys²⁸⁷ in human DHS). The mismatch created by the GRON produces a smalldeletion, insertion or single-base pair substitution in a targetedregion of plant DHS that is known to be critical for its deoxyhypusinesynthase activity. In M. sativa, the activity of the enzyme is disruptedby deleting one or more of the following residues: Lys³³⁴ (correspondsto Lys³²⁹ of human DHS—see Table 3) or Lys²⁹² (Lys²⁸⁷ in human DHS). Forexample, the nucleotide region encoding the region surrounding, andincluding, Lys³³⁴ of M. sativa DHS is deleted in order to abolishdeoxyhypusine synthase activity. In another example, the nucleotideregion encoding the region surrounding, and including, Lys²⁹² of M.sativa DHS is deleted in order to abolish deoxyhypusine synthaseactivity.

Example 6: Editing Pre-Determined Genomic Loci in alfalfa (Medicagosativa)

One or more gRNAs is designed to anneal with a desired site in thealfalfa genome and to allow for interaction with one or more Cms1 orother CRISPR double stranded nuclease proteins. These gRNAs are clonedin a vector such that they are operably linked to a promoter that isoperable in a plant cell (the gRNA cassette). One or more genes encodinga Cms1 or other CRISPR double stranded nuclease protein is cloned in avector such that they are operably linked to a promoter that is operablein a plant cell (the CRISPR nuclease cassette). The gRNA cassette andthe CRISPR nuclease cassette are each cloned into a vector that issuitable for plant transformation, and this vector is subsequentlytransformed into Agrobacterium cells. These cells are brought intocontact with alfalfa tissue that is suitable for transformation.Following this incubation with the Agrobacterium cells, the alfalfacells are cultured on a tissue culture medium that is suitable forregeneration of intact plants with selection against Agrobacteriumcells. Alfalfa plants are regenerated from the cells that were broughtinto contact with Agrobacterium cells harboring the vector thatcontained the CRISPR nuclease cassette and gRNA cassette. Followingregeneration of the alfalfa plants, plant tissue is harvested and DNA isextracted from the tissue. T7 endonuclease 1 (T7E1) assays and/orsequencing assays are performed, as appropriate, to determine whether achange in the DNA sequence has occurred at the desired genomic location.

Alternatively, particle bombardment is used to introduce the CRISPRnuclease cassette and gRNA cassette into alfalfa cells. Vectorscontaining a CRISPR nuclease cassette and a gRNA cassette are coatedonto gold beads or titanium beads that are then used to bombard alfalfatissue that is suitable for regeneration. Following bombardment, thealfalfa tissue is transferred to tissue culture medium for regenerationof alfalfa plants. Following regeneration of the alfalfa plants, planttissue is harvested and DNA is extracted from the tissue. T7EI assaysand/or sequencing assays are performed, as appropriate, to determinewhether a change in the DNA sequence has occurred at the desired genomiclocation.

Example 7: Editing Pre-Determined Genomic Loci in Oryza sativa

One or more gRNAs is designed to anneal with a desired site in the Oryzasativa genome and to allow for interaction with one or more Cms1 orother CRISPR double stranded nuclease proteins. These gRNAs are clonedin a vector such that they are operably linked to a promoter that isoperable in a plant cell (the gRNA cassette). One or more genes encodinga Cms1 or other CRISPR double stranded nuclease protein is cloned in avector such that they are operably linked to a promoter that is operablein a plant cell (the CRISPR nucleasecassette). The gRNA cassette and theCRISPR nucleasecassette are each cloned into a vector that is suitablefor plant transformation, and this vector is subsequently transformedinto Agrobacterium cells. These cells are brought into contact withOryza sativa tissue that is suitable for transformation with selectionagainst Agrobacterium cells. Following this incubation with theAgrobacterium cells, the Oryza sativa cells are cultured on a tissueculture medium that is suitable for regeneration of intact plants. Oryzasativa plants are regenerated from the cells that were brought intocontact with Agrobacterium cells harboring the vector that contained theCRISPR nuclease cassette and gRNA cassette. Following regeneration ofthe Oryza sativa plants, plant tissue is harvested and DNA is extractedfrom the tissue. T7EI assays and/or sequencing assays are performed, asappropriate, to determine whether a change in the DNA sequence hasoccurred at the desired genomic location.

Alternatively, particle bombardment is used to introduce the CRISPRnuclease cassette and gRNA cassette into Oryza sativa cells. Vectorscontaining a CRISPR nucleasecassette and a gRNA cassette are coated ontogold beads or titanium beads that are then used to bombard Oryza sativatissue that is suitable for regeneration. Following bombardment, theOryza sativa tissue is transferred to tissue culture medium forregeneration of intact plants. Following regeneration of the plants,plant tissue is harvested and DNA is extracted from this tissue. T7EIassays and/or sequencing assays are performed, as appropriate, todetermine whether a change in the DNA sequence has occurred at thedesired genomic location.

Example 8: Editing Pre-Determined Genomic Loci in Brassica napus

One or more gRNAs is designed to anneal with a desired site in theBrassica napus genome and to allow for interaction with one or more Cms1or other CRISPR double stranded nuclease proteins. These gRNAs arecloned in a vector such that they are operably linked to a promoter thatis operable in a plant cell (the gRNA cassette). One or more genesencoding a Cms1 or other CRISPR double stranded nuclease protein iscloned in a vector such that they are operably linked to a promoter thatis operable in a plant cell (the CRISPR nucleasecassette). The gRNAcassette and the CRISPR nucleasecassette are each cloned into a vectorthat is suitable for plant transformation, and this vector issubsequently transformed into Agrobacterium cells. These cells arebrought into contact with Brassica napus tissue that is suitable fortransformation. Following this incubation with the Agrobacterium cells,the Brassica napus cells are cultured on a tissue culture medium that issuitable for regeneration of intact plants with selection againstAgrobacterium cells. Brassica napus plants are regenerated from thecells that were brought into contact with Agrobacterium cells harboringthe vector that contained the CRISPR nuclease cassette and gRNAcassette. Following regeneration of the Brassica napus plants, planttissue is harvested and DNA is extracted from the tissue. T7EI assaysand/or sequencing assays are performed, as appropriate, to determinewhether a change in the DNA sequence has occurred at the desired genomiclocation.

Alternatively, particle bombardment is used to introduce the CRISPRnuclease cassette and gRNA cassette into Brassica napus cells. Vectorscontaining a CRISPR nuclease cassette and a gRNA cassette are coatedonto gold beads or titanium beads that are then used to bombard Brassicanapus tissue that is suitable for regeneration. Following bombardment,the Brassica napus tissue is transferred to tissue culture medium forregeneration of intact plants. Following regeneration of the plants,plant tissue is harvested and DNA is extracted from this tissue. T7EIassays and/or sequencing assays are performed, as appropriate, todetermine whether a change in the DNA sequence has occurred at thedesired genomic location.

Example 9: Deleting DNA from a Pre-Determined Genomic Locus Using NonHomologous End Joining

A first gRNA is designed to anneal with a first desired site in thegenome of a plant of interest and to allow for interaction with one ormore Cms1 or other CRISPR double stranded nuclease proteins. A secondgRNA is designed to anneal with a second desired site in the genome of aplant of interest and to allow for interaction with one or more CRISPRnuclease proteins. Each of these gRNAs is operably linked to a promoterthat is operable in a plant cell and is subsequently cloned into avector that is suitable for plant transformation. One or more genesencoding a Cms1 or other CRISPR double stranded nuclease protein iscloned in a vector such that they are operably linked to a promoter thatis operable in a plant cell (the “CRISPR nuclease cassette”). The CRISPRnuclease cassette and the gRNA cassettes are cloned into a single planttransformation vector that is subsequently transformed intoAgrobacterium cells. These cells are brought into contact with planttissue that is suitable for transformation. Following this incubationwith the Agrobacterium cells, the plant cells are cultured on a tissueculture medium that is suitable for regeneration of intact plants withselection against Agrobacterium cells. Alternatively, the vectorcontaining the CRISPR nuclease cassette and the gRNA cassettes is coatedonto gold or titanium beads suitable for bombardment of plant cells. Thecells are bombarded and are then transferred to tissue culture mediumthat is suitable for the regeneration of intact plants. The gRNA-CRISPRnuclease complexes effect double-stranded breaks at the desired genomicloci and in some cases the DNA repair machinery causes the DNA to berepaired in such a way that some native DNA sequence that was locatednear or within the gRNA sequence is deleted. Plants are regenerated fromthe cells that are brought into contact with Agrobacterium cellsharboring the vector that contains the CRISPR nuclease cassette and gRNAcassettes or are bombarded with beads coated with this vector. Followingregeneration of the plants, plant tissue is harvested and DNA isextracted from the tissue. T7EI assays and/or sequencing assays areperformed, as appropriate, to determine whether DNA has been deletedfrom the desired genomic location or locations.

Example 10: Making Base Substitutions in DNA at a Pre-Determined GenomicLocus Using Homology Directed Repair

A gRNA is designed to anneal with a desired site in the genome of aplant of interest and to allow for interaction with one or more Cms1 orother CRISPR double stranded nuclease proteins. The gRNA is operablylinked to a promoter that is operable in a plant cell and issubsequently cloned into a vector that is suitable for planttransformation. One or more genes encoding a Cms1 or other CRISPR doublestranded nuclease protein is cloned in a vector such that they areoperably linked to a promoter that is operable in a plant cell (theCRISPR nuclease cassette), along with a highly expressed single ordouble stranded donor DNA oligonucleotide comprised of a sequencehomologous to a targeted DNA in the host genome but containing specificbase changes that cause one or more targeted mutations that occur byHomology Directed Repair [Miki et al. (2018) Nature Comm. 9:1967-1975].The CRISPR nuclease cassette, the gRNA cassette, and the highlyexpressed oligonucleotide are cloned into a single plant transformationvector that is subsequently transformed into Agrobacterium cells. Thesecells are brought into contact with plant tissue that is suitable fortransformation. Said donor DNA oligonucleotide includes a DNA moleculethat is to be inserted at the desired site in the plant genome, flankedby upstream and downstream flanking regions. The upstream flankingregion is homologous to the region of genomic DNA upstream of thegenomic locus targeted by the gRNA, and the downstream flanking regionis homologous to the region of genomic DNA downstream of the genomiclocus targeted by the gRNA. The upstream and downstream flanking regionsmediate the recombination of DNA into the desired site of the plantgenome. Following this incubation with the Agrobacterium cells andintroduction of the donor DNA, the plant cells are cultured on a tissueculture medium that is suitable for regeneration of intact plants withselection against Agrobacterium cells. Plants are regenerated from thecells that were brought into contact with Agrobacterium cells harboringthe vector that contained the CRISPR nuclease cassette, gRNA cassettesand donor DNA oligonucleotide. Following regeneration of the plants,plant tissue is harvested and DNA is extracted from the tissue. T7EIassays and/or sequencing assays are performed, as appropriate, todetermine whether the desired base changes have occurred at the desiredgenomic location or locations.

Example 11: DHS Editing in Brassica napus Using CRISPR-Cas9

Brassica napus (Canola) is an important oilseed crop and a dicot. Theamphidiploid genome of B. napus has two copies of the DHS gene, one eachfrom the contributing B. rapa (AA) and B. oleracea (CC) genomes. TheLOC106419026 DHS gene (DHS1) of B. napus was edited using theCRISPR-Cas9 system to determine if senescence could be delayed. Themethods for this example were adapted from previous gene editing studiesin B. napus, which are herein incorporated in their entirety [Yang etal., Scientific Reports 7:7489, 2017].

Vector construction: The DNA sequences of the B. napus DHS gene thatwere selected for targeting by guide RNAs are identified FIG. 4. A plantdelivery vector comprising the three guide RNA sequences was constructedin three steps according to the method of Lowder [Lowder et al., PlantPhysiology 169:971-985, 2015].

Step 1) To reduce subcloning background, the AtU6-based cassette donorvector was digested first with Bgl II and Sal I restrictionendonucleases, followed by a second digestion with Esp3I per thepublished protocol for assembly of a multiplex CRISPR-Cas9 T-DNA vector(www.plantphysiol.org/content/plantphysiol/suppl/2015/08/21/pp.15.00636.DC1/PP2015-00636R1_Supplemental_Materials_andmethods.pdf). Sense and antisense oligonucleotides with sequencescorresponding to the three DHS guide RNAs were synthesized, annealed,and then individually inserted into donor vectors. The sequences of theresulting plasmids, pYPQ131A, pYPQ132A and pYPQ133A, were confirmed bySanger sequencing.

Step 2) All three guide sequences were assembled into pYPQ143 using theGolden Gate® recombination system. Insertion of a Cas9 expression moduleinto pYPQ143 yielded recombinant plasmid vector pYPQ150, which containedall CRISPR components required for targeted editing of the B. napus DHSgene.

Step 3) The CRISPR cassettes were incorporated into a plant deliveryvector using the Gateway® recombination system to create a binarydestination plasmid that contains a third module with PAT and kanamycinselection markers for recovering the transformants. The sequences of theCRISPR cassettes in the transformation vector were confirmed by Sangersequencing. This derivative recombinant binary vector was introducedinto Agrobacterium MP90 carrying a disarmed Ti plasmid with virfunctions provided in trans.

Production of transgenic lines: The recombinant T-DNA was delivered intoreceptive B. napus cells. Cotyledonary petioles were collected from 4-5days old B. napus seedlings grown in jars under sterile conditions andexpanded in tissue culture [Moloney et al., Plant Cell Reports 8:238-42,1989; Babic et al., Plant Cell Reports 17:183-88, 1998]. Thecotyledonary explants were placed on filter paper and co-cultivated withAgrobacterium carrying the CRISPR-based DHS targeting construct inmedium containing 1 mg/l 2,4-Dichlorophenoxyacetic acid (2-4 D) for 2days at 22° C. followed by 4 days at 4° C. The explants were thentransferred to selection medium (MS) with 4 mg/l 6-benzyl aminopurine(BAP), 25 mg/l Kanamycin and 2.5 mg/l PPT. After 4-6 weeks, some of theexplants produced green shoots from the petioles on the selectionmedium. To increase the number of putative transformed shoots, thesesteps were repeated 4 times using ˜700 explants in each experiment.Green shoots recovered from these experiments were transferred to“elongation medium” containing 0.05 mg/l BAP and 0.02 mg/l GibberellinA3 (GA3). Shoots that were not vitrified were transferred to rootingmedium containing 50% MS plus 0.1 mg/l BAP, 25 mg/l kanamycin and 5 mg/lphosphinotricin (PPT). Shoots that successfully produced roots weretransferred to pots to establish T0 plants, which were analysed by PCRfor the presence of transgenes. A total of 55 B. napus transgenic lineswere identified. The stringent double selection and confirmativemolecular analysis ensured that these transgenic lines carry therecombinant DHS constructs. The DHS genes from 38 of these lines wereamplified by PCR, subcloned into a plasmid, and then sequenced. All butone were identical to wild type. The exception, clone #16, had a 1 bpdeletion adjacent to the sequence targeted by a guide RNA, resulting ina truncated DHS protein (FIG. 4). This truncated allele is missing theC-terminal active site of DHS and is expected to be completely inactive.Clone #16 also contained a wild-type DHS gene, indicating that it is aheterozygote.

Phenotype resulting from an edited B. napus DHS gene: The transgenic DHSlines were grown in pots under controlled greenhouse conditions at 22°C. with a 16 h light/8 hrs dark cycle. Their development and growth wascompared with non-transgenic control lines. The majority of thetransgenic lines had DHS gene sequences identical to wild type and hadnormal growth and development patterns. In contrast, B. napus line #16,with an edited DHS gene, produced dwarf plants with darker leaves. Line#16 also displayed delayed leaf senescence and took 15-20 days longer tocomplete the growth and seed production cycle (FIG. 5).

Beneficial effects of editing the B. napus DHS gene: The compact dwarfphenotype, darker green leaves, and delayed senescence of B. napusplants with an edited DHS gene offer agronomic advantages. The compactdwarf phenotype offers less lodging and better shoot architecture forcapturing sunlight. This could improve photosynthetic efficiency,thereby increasing oil production and seed yields in this important oilseed crop. The darker green leaves suggest a higher chlorophyll contentand increased photosynthetic capacity. Delayed senescence results in“staying green longer,” especially during critical and final phases ofreproductive development, and is expected to enhance seed filling andproduction in B. napus, as observed in other diverse crop species.

Example 12: DHS1 Editing in Japonica Rice Using CRISPR-Cms1 with a GuideRNA Targeting the Intron Upstream of K149

Oryza sativa cv. Kitaake (Japonica rice) is a monocot. Edits weregenerated in the intron upstream of the NAD⁺-binding site adjacent toK149 of the DHS1 gene of O. sativa cv. Kitaake. Most resulted in 3-21 bpdeletions that do not affect the exon sequence, but may causemis-splicing of the mRNA. Several larger (>50 nt) deletions and deletionwith insertions were also observed. These would be predicted to affectthe protein product, potentially resulting in a loss of function. T0events generated with larger modifications were compared to T0 eventsgenerated at the same time with smaller modifications. The largermodifications were associated with a delay in flowering and an increasein chlorophyll content.

Guide RNA design: Kitaake rice has a highly-expressed DHS1 gene(OsKitaake 03g332300 (DHS1) on Chr3 at 31549616-31554176) and ahomologous but rarely-expressed DHS2 gene (OsKitaake 09g102400 (DHS2) onChr9 at 14897349-14900826). A guide RNA specific to the DHS1 gene andcompatible with (CRISPR-Cms1 chemistry was designed to target the intronbetween the second and third exons. It had the following sequence,

[SEQ ID NO: 532] AATTTCTACTGTTGTAGATAAGGGGGATTAGCTACATCATAGG,with underlining indicating a crRNA hairpin structure.

Production of gene edited rice plants: Callus derived from immature O.sativa cv. Kitaake seeds was bombarded with three plasmids separatelycontaining a CRISPR nuclease, a guide RNA, and a marker providingresistance to hygromycin. After four weeks of selection, the callusmaterial was screened for the presence of edits with a T7 Enoduclease Iassay. Next Generation Sequencing of DNA from callus material withapparent edits revealed deletions at the target site ranging from 3 to65 base pairs (FIG. 6). Plants were regenerated from callus pieceshaving internal deletions in DHS1. The T0 plants were screened with theT7 exonuclease assay and sequenced for verification (FIGS. 7 and 8).Lines C31G, C31H, and C31I have larger insertions. DNA from line C31Hwas sequenced and found to have the same insertion/deletion as eventC30S, suggesting these two lines were derived from a common editingevent.

-   -   Phenotype of T0 rice plants: T0 rice plants were regenerated,        grown under greenhouse conditions, and compared with other T0        plants regenerated from the same callus material and        transplanted to soil on the same date. Chlorophyll content was        measured with a SPAD 502 Plus Chlorophyll Meter        (www.specmeters.com/nutrient-management/chlorophyll-meters/chlorophyll/spad502p/#description).        Lines C30S, C31G, C31H, and C31I all had a 10-day delay in        flowering relative and a >11% increase in chlorophyll content        compared to line C30T, indicating that large insertion/deletions        cause delayed flowering and increased chlorophyll content (Table        4). No significant phenotypic differences were observed between        lines derived from a separate callus piece (GE568-8) that had        either small internal deletions in the intron or no detected        edits (Table 5).

TABLE 4 Phenotypes of Kitaake Rice GE0568 T0 plants regenerated fromcallus 30 and 31. Chlorophyll Days content to (average SPAD T0 plant IDEdit Reproduction reading) GE0568 30T 3 bp deletion 21 32.9 GE0568 30S10 bp deletion & 31 36.6 99 bp insertion GE0568 31G Insertion 31 39.9GE0568 31H 10 bp deletion & 31 38.1 99 bp insertion GE0568 31I Insertion31 39.15

TABLE 5 Phenotypes of Kitaake Rice GE0568 T0 plants regenerated fromcallus 8. Chlorophyll Days content to (average SPAD T0 plant ID EditReproduction reading) GE0568 8W Small indel 28 36 GE0568 8S None 31 42.8GE0568 8T None 28 42.8 GE0568 8U None 28 42.8 GE0568 8X None 31 34.85

To confirm the phenotypic differences observed with T0 rice plants, T1plants are generated and genotyped by DNA sequencing. For each DNAediting event heterozygous plants are compared to null segregant andwild-type controls. In the unlikely event that homozygous mutant plantssurvive, they are also tested. For each genotype, twelve plants aregrown in a greenhouse and phenotyped for biomass, flowering time, daysto senescence, chlorophyll content and seed yield.

Example 13: DHS1 Editing in Japonica Rice Using CRISPR-Cms1 with a GuideRNA Targeting the Active Site Lysine

Guide RNA: A guide RNA [SEQ ID NO: 581] was designed to introduce amutation in the conserved active site (EAVSWGK; SEQ ID NO: 546) in exon6 of the Oryza sativa cv. Kitaake DHS1 gene, as illustrated in FIG. 9.Sequence corresponding to the guide RNA was inserted behind a rice U6small nuclear gene promoter in a plant delivery vector. The vectorfurther comprises a Cms1 gene under a maize ubiquitin gene promoter, anda hygromycin-resistance marker.

Production of gene edited rice plants: The plant delivery vector wastransferred into Agrobacterium for transformation of Kitaake rice. Sevenhygromycin-resistant T0 plants were regenerated and genotyped formutations at the targeted site. One T0 plant (#2) was a monoallelic (orheterozygous) mutant with one wild-type allele and the other allelecarrying a 10 bp deletion at the targeted active site. Other T0 plantswere either wild type (homozygous normal) or too weak to survive forgenotyping (homozygous mutant). Sequencing DNA extracted from plant #2revealed a 10 bp deletion resulting in a frameshift that leads to a stopcodon (FIG. 10; SEQ ID NO: 579). The resulting protein, missing 4 aminoacids from the conserved active site and the subsequent 37 amino acidsand is expected to be enzymatically inactive due to removal of 11% ofthe protein's total amino acids, yielding a protein of 334 amino acidsinstead of the wild-type 375 amino acids.

Phenotype of rice plants carrying C-terminally truncated DHS1: Theheterozygous T0 line #2 grew normally in the greenhouse with no apparentmorphological differences other than a significant increase in seed sizecompared to homozygous wild type plants. The T1 progeny included fourheterozygotes and six homozygous wild-types, but no homozygous DHS1truncation mutants, suggesting that the homozygous truncation is lethal.T1 plants were grown to maturity in the greenhouse. Compared towild-type controls, the heterozygous T1 plants are shorter, have aslightly higher tiller number, a similar spikelet number, and have seedthat are about 25% larger (Table 6). Additionally, T1 plants carryingthe C-terminally truncated DHS1 allele have a darker green appearance,suggesting higher chlorophyll content (FIG. 11).

The phenotypes observed in rice with a heterozygous C-terminallytruncated DHS1 allele demonstrate the benficial effects of reducing DHSactivity through introducing specific mutations or deletions in genomeedited crops, consistent with previous observations of reducing DHSexpression using RNAi in Arabidopsi thaliana, tomato, and canola usingantisense RNA [Duguay 2007; Wang 2001; Wang et al. (2003) Plant Mol.Biol. 52, 1223-1235; Wang et al. (2005) Physiol. Plant. 124, 493-503;Wang et al. (2005) Plant Physiol. 138, 1372-1382]. A 50% reduction inDHS activity using genome editing methods to make critical mutationsseems to be in the “sweet spot” that these authors were able to obtainusing very different transgenic antisense derivatives of wild-type DHSin several crops. DHS assembles into tetramers, so a reduction inactivity might be caused by the formation of tetramers with a mix offull-length (active) and C-terminally truncated (inactive) subunits,segregation of full-length and C-terminally truncated DHS polypeptidesinto separate tetramers, or the premature degradation of C-terminallytruncated polypeptides.

TABLE 6 Phenotypic comparison of T1 rice plants. Values are presented asmean ± standard deviation. Trait Truncated DHS Control Height (cm) 56.8± 16.6 69.4 ± 3.8  Tiller number 8.0 ± 2.6 6.0 ± 1.7 Spikelet number36.7 ± 9.1  37.2 ± 5.5  Seed mass (g/20 seeds) 0.75 ± 0.03 0.59 ± 0.02

Example 14: General Strategy for Base Editing DHS in Various Plants

DHS activity is disrupted by genome or base editing the NAD⁺-bindingsite adjacent to Lys₁₄₉ and/or the active site near the C-terminus.Lys₁₄₉ can be edited by replacing the second adenine in a Lys codon witha guanine to form an Arg codon, or by deletion. The 21 nucleotidesencoding the active site can be altered by a specific mutation ordeleted as part of a C-terminal truncation (Table 7).

TABLE 7Site-Specific codon changes to NAD⁺-binding sites near Lys₁₄₉ in genomic Exon 3/4 of DHS genes and21-NT active site sequences. Active SEQ Species Lys₁₄₉ Lys₁₄₉  Arg SiteSequence coding for the ID ID Species name Common name Exon codon codonExon 7-amino acid Active Site NO Adur Arachis duranensis peanut parent 14 AAG AGG 7 GAAGCTGTTTCCTGGGGAAAA 547 Aipa Arachis ipaensispeanut parent 2 4 AAG AGG 7 GAAGCTGTTTCCTGGGGAAAA 548 AthaArabidopsis thaliana Arabidopsis 3 AAA AGA 6 GAAGCCGTGTCTTGGGGTAAA 549Bnap Brassica napus canola 3 AAA AGA 6 GAAGCAGTGTCTTGGGGTAAA 550 BrapBrassica rapa rape 3 AAA AGA 6 GAGGCAGTGTCTTGGGGTAAA 551 Bvu1Beta vulgaris subsp. sugarbeet 3 AAA AGA 6 GAGGCCGTGTCCTGGGGAAAG 552vulgaris Cari Cicer arietinum chickpea 3 AAG AGG 6 GAGGCTGTTTCATGGGGGAAA553 Ccan Coffea canephora coffee 3 AAG AGG 6 GAAGCTGTATCATGGGGAAAG 554Csat Camelina sativa camelina 3 AAA AGA 6 GAAGCCGTATCTTGGGGTAAA 555 CsinCamellia sinensis tea 3 AAA AGA 6 GAAGCTGTATCATGGGGAAAA 556 GhirGossypium hirsutum cotton 4 AAA AGA 7 GAAGCTGTTTCATGGGGGAAA 557 Gmax1Glycine max soybean 4 AAG AGG 7 GAAGCTGTTTCATGGGGAAAG 558 Gmax2Glycine max soybean 4 AAG AGG 7 GAAGCTGTTTCATGGGGAAAG 559 HsapHomo sapiens human 3 AAG — 8 GAGGCTGTCTCCTGGGGCAAG 560 LsatLactuca sativa lettuce 3 AAA AGA 6 GAAGCTGTCTCCTGGGGGAAA 561 MacuMusa acuminate banana 3 AAA AGA 6 GAGGCGATTTCATGGGGAAAG 562 MescManihot esculenta cassava 4 AAA AGA 7 GAGGCTGTATCATGGGGAAAA 563 MsatMedicago sativa alfalfa 4 AAG AGG 7 GAGGCTGTTTCATGGGGGAAA 564 OsatOryza sativa Japonica rice 3 AAA AGA 6 GAAGCAGTTTCATGGGGAAAG 565 Pde1Populus deltoides cottonwood 3 AAA AGA 6 GAGGCTGTATCGTGGGGGAAA 566 PequPhalaenopsis equestris orchid 3 AAG AGG 6 GAGGCTGTTTCATGGGGAAAA 567 Pvu1Phaseolus vulgaris common bean 3 AAG AGG 6 GAGGCTGTTTCGTGGGGGAAA 568Rchi1 Rosa chinensis rose 3 AAA AGA 6 GAGGCTGTCTCCTGGGGGAAA 569 Rchi2Rosa chinensis rose 3 AAA AGA 6 GAGGCTGTCTCCTGGGGGAAA 570 SbicSorghum bicolor sorghum 3 AAA AGA 6 GAAGCAGTCTCATGGGGCAAG 571 SlycSolanum lycopersicum tomato 3 AAG AGG 6 GAAGCTGTATCATGGGGAAAG 572 StubSolanum tuberosum potato 3 AAG AGG 6 GAAGCTGTATCATGGGGAAAG 573 TaesTriticum aestivum wheat 3 AAA AGA 6 GAAGCAGTTTCATGGGGAAAG 574 TcacTheobroma cacao cocoa 4 AAG AGG 6 GAGGCTATTTCATGGGGGAAA 575 VvinVitis vinifera grape 3 AAA AGA 6 GAGGCTGTGTCATGGGGGAAA 576 Zmay1Zea mays corn 3 (5) AAA AGA 6(8) GAAGCAGTCTCATGGGGCAAG 577 Zmay2Zea mays corn 3 (6) AAA AGA 6(9) GAAGCGGTTTCATGGGGAAAG 578

1. A method of producing a plant with delayed senescence comprisinginducing at least one nucleotide deletion, insertion or substitutioninto at least one copy of a gene encoding deoxyhypusine synthase (DHS)in the plant, wherein the nucleotide deletion, insertion or substitutiondecreases the activity of DHS encoded by the gene in the plant.
 2. Themethod of claim 1, wherein the nucleotide deletion, insertion orsubstitution occurs in a coding region for at least one amino acid whichis required for DHS activity.
 3. The method of claim 2, wherein the DHSactivity is hypusination of eukaryotic translation initiation factor 5A(eIF-5A) in the plant.
 4. The method of claim 1, wherein the nucleotidedeletion, insertion or substitution decreases the activity of DHSencoded by the gene in the plant.
 5. The method of claim 1, wherein thedelayed senescence increases seed yield in the plant.
 6. The method ofclaim 1, wherein the delayed senescence increases leaf and root biomass.7. The method of claim 1, wherein the delayed senescence enhances plantsurvival during drought or nutrient stress.
 8. The method of claim 1,wherein the delayed senescence increases disease resistance of theplant.
 9. The method of claim 1, wherein the delayed senescenceincreases the period of time during which leaves, stems, seeds and fruitof the plant may be stored and remain suitable for use.
 10. The methodof claim 1, wherein the plant is haploid, diploid, tetraploid orpolyploid.
 11. The method of claim 1, comprising inducing at least onenucleotide deletion, insertion or substitution into at least two copiesof a gene encoding DHS in the plant. 12-61. (canceled)
 62. The method ofclaim 1, wherein the deletion, insertion or substitution into at leastone copy of a gene is in a coding region for at least one amino acidselected from the group consisting of K334, K292, K343, E328, A329,V330, S331, W332, G333, K144, D318 of SEQ ID NO: 2 or a correspondingamino acid in a related plant species.
 63. The method of claim 1,wherein the senescence is age-related senescence.
 64. The method ofclaim 1, wherein the senescence is environmental stress-inducedsenescence.
 65. A plant produced by the method of claim
 1. 66. Progenyof the plant according to claim 65.